To help define the physiological targets of general anesthetics, we are isolating mutations of Drosophila melanogaster that alter the effectiveness of these agents. In the past both new and existing mutants have been characterized by a simple behavioral assay of the fly's capacity to retain an upright posture and by a more sophisticated electrophysiological analysis of an escape reflex. To supplement our repertory of behavioral assays we have now developed a "distribution" test that measures the fly's capacity to climb up the side of a tube after being shaken to its bottom. This test provides a rapid and sensitive assay that can be performed reliably with a smaller number of individuals than our other assays. Mutants isolated as resistant to the effects of halothane by the postural test are hypersensitive to halothane in the distribution test, confirming that these mutations do not eliminate the principal target of halothane in the organism. To refine our electrophysiological analysis, we have redesigned the setup to reduce the flow of anesthetic by a factor of approximately 1,000. This will permit studies with research anesthetics that are available only in limited quantity. We have also used this setup to test the effect of halothane on different wild-type strains of Drosophila. Surprisingly, we find that substantial differences in sensitivity exist between these lines. We conclude that studies of mutants will have to be exquisitely controlled for genetic background. Finally, we have tested the escape response by behavioral, as opposed to electrophysiological, methods in free-ranging flies. The sensitivity to halothane in both assays is comparable, confirming the value of the escape response as a tool for identifying physiological targets of this agent.